Guidelines
CELL LINES
PLANT CELL LINES
PLANT VIRUSES
PLASMIDS
PHAGES
MICROORGANISMS
GENOMIC LIBRARIES
PROCEDURES
CATALOGUE
LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/3
TITLE: QUALITY CONTROLS
Contamination of cell cultures can lead to an array of problems, many of which can be underestimated. The damage caused to cell lines in culture by bacteria and fungi contamination is widely recognised and normally easy to see. In contract the effects of mycoplasma can be wide ranging and less obvious including: a reduction in growth rate, induction of morphological changes, chromosomal aberrations altering cell metabolism and loss of cell recovery following cryopreservation.
All the cell lines proposed by CABRI are tested to verify freedom from contamination by mycoplasma, bacteria, yeast, fungi and virus. In addition, the cell lines undergone tests to confirm species of origin and tests for individual identification.
FLOW DIAGRAM GENERAL CONSIDERATIONS ON MYCOPLASMA
Minimum QC requirements
(
A) Preliminary ScreenViability Check
- Cell count and viability:
- vital dye exclusion (trypan blue)
Acceptance criteria: > 2 x 106 cells/vial, > 80% viability, vigorous cell growth
Microbial QC
- Mycoplasma testing
- Hoechst stain/DAPI stain,
- Isolation by culture
- Bacteria/fungi testing
- Isolation by culture,
Acceptance criteria - all tests negative
(B) Full QC and Authentication Screen Master cell banks
Viability Check
- Cell count and viability:
- vital dye exclusion (trypan blue), AHC/1998/3/3.1/2.1
Acceptance criteria - > 2 x 106 cells/vial, > 80% viability, vigorous cell growth
Microbial QC
- Mycoplasma testing
- Hoechst stain/DAPI stain, AHC/1998/3/3.2/2.1
- Isolation by culture, AHC/1998/3/3.2/2.2
- Bacteria/fungi testing
- Isolation by culture, AHC/1998/3/3.3/2.1
Acceptance criteria - all tests negative
Authentication Testing
- Isoenzyme analysis
Acceptance criteria - species confirmed as correct
Additional testing is carried out at individual centres as indicated below.
Authentication Assays
- DNA fingerprinting (DSMZ, ECACC) - AHC/1998/3/3.1/2.3
- Chromosome analysis (DSMZ) - AHC/1998/3/3.1/1.1
- Antibody production of hybridomas (ECACC) - AHC/1998/3/3.5/2
- Immunophenotyping of tumour lines (DSMZ) - AHC/1997/3/3.1/1.4
Microbial QC
- DNA-RNA hybridisation (DSMZ)
- PCR detection of mycoplasma (INRC) -
Virus Screening
- PCR analysis for HIV (DSMZ, ECACC) - AHC/1998/3/3.4/1.1
- PCR analysis for HTLV 1,2 (DSMZ, ECACC) - AHC/1998/3/3.4/1.2
- PCR analysis for BVDV (ECACC) -
(C) Full QC and Authentication Screen Distribution cell banks
Viability Check
- Cell count and viability:
- vital dye exclusion (trypan blue),
Acceptance criteria - > 2 x 106 cells/vial, > 80% viability, vigorous cell growth
Microbial QC
- Mycoplasma testing
- Hoechst stain/DAPI stain: AHC/1998/3/3.2/2.1
- Isolation by culture
- Bacteria/fungi testing
- Isolation by culture:
Acceptance criteria - all tests negative
Additional testing is carried out at individual centres as indicated below.
Authentication Assays
- DNA fingerprinting (DSMZ, ECACC) -
Microbial QC
- DNA-RNA hybridisation (DSMZ)
- PCR detection of mycoplasma (INRC) -
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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Copyright CABRI, 1998
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